1. Field of the Invention
This invention relates to an assay applicable to clinical samples for an enzyme which catalyses directly or indirectly the release of long chain fatty acids or, conversely, for an ester substrate for such enzymes. The invention is particularly, though not exclusively, directed to the assay of lipases and phospholipases or for the ester substrates for such enzymes such as the triglyceride substrates for lipase and phospholipid substrates for the phospholipases.
2. Description of the Prior Art
Lipase, phospholipases and substrates therefor, such as the tri and diglycerides are ubiquitous and fundamental to every aspect of cell membrane function and energy transfer. The assay of these components is therefore of interest in many areas of clinical diagnosis. Thus, for example, assay of the triglyceride content of a clinical specimen can give some indication of recent dietary fat intake, and the ability of the liver to metabolise fats for energy utilisation. However presently available methods for assay of triglycerides, such as those available commercially as kits from the Sigma Chemical Co. Ltd. (Poole, Dorset, UK) rely on the measurement of glycerol released by enzyme hydrolysis. Thus, in Sigma procedure no. 405, triglycerides are extracted into isopropanol and saponified with potassium hydroxide. Liberated glycerol is then converted to formaldehyde by periodate. By reacting with acetylacetone, the formaldehyde forms yellow diacetyldihydrolutidine, which is measured colorimetrically.
In Sigma procedures nos. 336, 337, 339 and 334 glycerol is released from tryglyceride enzymatically using lipase, and glycerol is further reacted with ATP to form glycerol-1-phosphate. The four methods then differ only in the way by which the glycerol-1-phosphate is further reacted to produce a change in absorbance which can be measured spectrophotometrically. Such assays, when applied to clinical samples, suffer from the disadvantage that as glycerol itself is a product of cell metabolism, assay of the glycerol content if a blood specimen may not give an accurate picture of the circulating triglyceride levels in the subject (see Cole, Clin. Chem. 36/7, 1267-1268 (1960). There is clearly a need for the development of alternative means for assaying triglycerides that give accurate results at low concentrations or in small clinical blood specimens.
There is also a need for a rapid and accurate assay at low concentrations of lipase activity. Thus in clinical diagnosis, there is a need for the reliable detection of pancreatitis, particularly in relation to acute abdominal emergencies. Confirmation of a diagnosis of pancreatitis depends on the detection of elevated levels of pancreatic enzymes in the blood, particularly amylase and lipase, but this is normally achieved by measuring amylase levels. Amylase is not an ideal marker, because due to its small size it is partly filtered by the kidneys and levels in the pancreas may therefore be affected by kidney dysfunction. Elevated blood levels of amylase may also result from disorders of the parotid glands, which produce large amounts of salivary amylase. Lipase is potentially a better alternative, see M. Ventrucci et al., Digestive Diseases and Science 34, 39-45 (January 1989), which also contains references to assays of this kind. However, the clinical assays which have so far been developed for this enzyme are both slow and insensitive. Sigma procedure no. 800 for the measurement of lipase activity in serum is typical. Using olive oil as substrate for the enzyme, the quantity of fatty acids formed is measured by titration with dilute standardised sodium hydroxide solution. The quantity of alkali required to reach the thymolphthalein endpoint is proportional to lipase activity.
This method is clearly not suitable for use as an emergency clinical procedure. A rapid and sensitive assay for serum lipase is therefore also required.